Antibacterial compounds

ABSTRACT

Compounds of the formula (I) are disclosed which are FabI inhibitors and are useful in the treatment bacterial infections:wherein:R1 is Ar or Het;R2 is H, C1-6alkyl or Ar-C0-6alkyl;X is H, C1-4alkyl, OR&#39;, SR&#39;, C1-4alkylsulfonyl, C1-4alkylsulfoxyl, CN, N(R&#39;)2, CH2N(R&#39;)2, NO2, CF3, CO2R&#39;, CON(R&#39;)2, COR&#39;, NR&#39;C(O)R&#39;, F, Cl, Br, I, or CF3S(O)r-;R&#39; is H, C1-6alkyl or Ar-C0-6alkyl; andr is 0, 1 or 2;or a pharmaceutically acceptable salt thereof.

This application is a 371 of PCT/US00/15154, filed on Jun. 1, 2000 whichclaims benefit of Ser. No. 60/137004, filed on Jun. 1, 1999.

FIELD OF THE INVENTION

This invention relates to pharmaceutically active compounds whichinhibit FabI and are useful for the treatment of bacterial infections.

BACKGROUND OF THE INVENTION

While the overall pathway of saturated fatty acid biosynthesis issimilar in all organisms, the fatty acid synthase (FAS) systems varyconsiderably with respect to their structural organization. Vertebratesand yeast possess a FAS in which all the enzymatic activities areencoded on one or two polypeptide chains, respectively, and the acylcarrier protein (ACP) is an integral part of the complex. In contrast,in bacterial FAS, each of the reactions is catalyzed by a distinct,mono-functional enzyme and the ACP is a discrete protein. Therefore,there is considerable potential for the selective inhibition of thebacterial system by antibacterial agents.

FabI (previously designated EnvM) functions as an enoyl-ACP reductase(Bergler, et al, (1994), J.Biol.Chem. 269, 5493-5496) in the final stepof the four reactions involved in each cycle of bacterial fatty acidbiosynthesis. In this pathway, the first step is catalyzed byβ-ketoacyl-ACP synthase, which condenses malonyl-ACP with acetyl-CoA(FabH, synthase III). In subsequent rounds, malonyl-ACP is condensedwith the growing-chain acyl-ACP (FabB and FabF, synthases I and II,respectively). The second step in the elongation cycle is ketoesterreduction by NADPH-dependent β-ketoacyl-ACP reductase (FabG). Subsequentdehydration by β-hydroxyacyl-ACP dehydrase (either FabA or FabZ) leadsto trans-2-enoyl-ACP, which in turn is converted to acyl-ACP byNADH-dependent enoyl-ACP reductase (FabI). Further rounds of this cycle,adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP(16C), where upon the cycle is stopped largely due to feedbackinhibition of FabI by palmitoyl-ACP (Heath, et al, (1996), J.Biol.Chem.271, 1833-1836). Thus, FabI is a major biosynthetic enzyme and is a keyregulatory point in the overall synthetic pathway of bacterial fattyacid biosynthesis. Therefore, FabI is an ideal target for antibacterialintervention.

Studies have shown that diazaborine antibiotics inhibit fatty acid,phospholipid and lipopolysaccharide (LPS) biosynthesis and that theantibacterial target of these compounds is FabI. For example, derivative2b18 from Grassberger, et al (1984) J. Med Chem 27 947-953 has beenreported to be a non-competitive inhibitor of FabI (Bergler, et al,(1994), J.Biol.Chem. 269, 5493-5496). Also, plasmids containing the FabIgene from diazaborine resistant S. typhimurium conferred diazaborineresistance in E.coli (Tumowsky, et al, (1989), J.Bacteriol., 171,6555-6565). Furthermore, inhibition of FabI either by diazaborine or byraising the temperature in a FabI temperature sensitive mutant islethal. These results demonstrate that FabI is essential to the survivalof the organism (Bergler, et al, (1994), J.Biol.Chem. 269, 5493-5496).

Recent studies have shown that FabI is also the target for the broadspectrum antibacterial agent triclosan (McMurry, et al, (1998) Nature394, 531-532). A crystal structure of the E. Coli FabI complexed withNAD and triclosan shows that triclosan acts as a site-directed, verypotent inhibitor of FabI by mimicking its natural substrate (Levy, etal, (1999) Nature 398, 383-384). Ward, et al ((1999) Biochem. 38,12514-12525) have shown that there is no evidence for the formation of acovalent complex between FabI and triclosan, which would be analogous tothe diazaborines; triclosan differs from these compounds in that it is areversible inhibitor of FabI. The structural data for the complex ofFabI with NAD and triclosan provides important information about FabI asa therapeutic target.

Importantly, it has now been discovered that certain compounds are FabIinhibitors and have antibacterial activity, and, therefore, may beuseful for the treatment of bacterial infections in mammals,particularly in man.

SUMMARY OF THE INVENTION

This invention comprises compounds of the formula (I), as describedhereinafter, which inhibit FabI and are useful in the treatment ofbacterial infections.

This invention is also a pharmaceutical composition comprising acompound according to formula (I) and a pharmaceutically acceptablecarrier.

This invention is also a method of treating bacterial infections byinhibiting FabI. In a particular aspect, the compounds of this inventionare useful as antibacterial agents.

DETAILED DESCRIPTION

This invention comprises compounds of formula (I):

wherein:

R¹ is Ar or Het;

R² is H, C₁₋₆alkyl or Ar—C₀₋₆alkyl;

X is H, C₁₋₄alkyl, OR′, SR′, C₁₋₄alkylsulfonyl, C₁₋₄alkylsulfoxyl, CN,N(R′)₂, CH₂N(R′)₂, NO₂, CF₃, CO₂R′, CON(R′)₂, COR′, NR′C(O)R′, F, Cl,Br, I, or CF₃S(O)_(r)—;

R′ H, C₁₋₆alkyl or Ar—C₀₋₆alkyl; and

r is 0, 1 or 2;

or a pharmaceutically acceptable salt thereof.

Also included in this invention are pharmaceutically acceptable additionsalts and complexes of the compounds of this invention. In cases whereinthe compounds of this invention may have one or more chiral centers,unless specified, this invention includes each unique racemic compound,as well as each unique nonracemic compound.

In cases in which compounds have unsaturated carbon-carbon double bonds,both the cis (Z) and trans (E) isomers are within the scope of thisinvention. In cases wherein

compounds may exist in tautomeric forms, such as keto-enol tautomers,such as

and

each tautomeric form is contemplated as being included within thisinvention, whether existing in equilibrium or locked in one form byappropriate substitution with R′. The meaning of any substituent at anyone occurrence is independent of its meaning, or any other substituent'smeaning, at any other occurrence.

Also included in this invention are prodrugs of the compounds of thisinvention. Prodrugs are considered to be any covalently bonded carrierswhich release the active parent drug according to formula (I) in vivo.

The compounds of formula (I) inhibit FabI. Inhibition of this enzyme isuseful in the treatment of bacterial infections. Also, the compounds ofthis invention may be useful as antifungal agents. Additionally, thecompounds may be useful in combination with known antibiotics.

With respect to formula (I):

Suitably, R¹ is phenyl, unsubstituted or substituted by methylenedioxyor by one to three substituents selected from the group consisting ofC₁₋₄alkyl, OR′, N(R′)₂, F, Cl, Br, I, and CF₃, in which R′ is H orC₁₋₆alkyl.

Alternately, R¹ is benzimidazolyl or pyridinyl, unsubstituted orsubstituted by C₁₋₄alkyl, OR′, N(R′)₂, F, Cl, Br, I, and CF₃, in whichR′ is H or C₁₋₆alkyl.

Suitably, R² is H, C₁₋₆alkyl or phenyl-C₀₋₆alkyl, wherein phenyl isunsubstituted or substituted by methylenedioxy or by one to threesubstituents selected from the group consisting of C₁₋₄alkyl, OR″,CO₂R′, N(R′)₂, F, Cl, Br, I, and CF₃, in which R′ is H or C₁₋₆alkyl andR″ is H, C₁₋₆alkyl or benzyl.

Suitably, X is H, C₁₋₄alkyl, OR′, CN, N(R′)₂, NO₂, CF₃, CO₂R′, CON(R′)₂,COR′, F, Cl, Br or I, in which R′ is H or C₁₋₆alkyl.

Representative of the novel compounds of this invention are thecompounds named in Examples 1-24 hereinafter.

Abbreviations and symbols commonly used in the peptide and chemical artsare used herein to describe the compounds of this invention. In general,the amino acid abbreviations follow the IUPAC-IUB Joint Commission onBiochemical Nomenclature as described in Eur J. Biochem., 158, 9 (1984).

C₁₋₄alkyl as applied herein means an optionally substituted alkyl groupof 1 to 4 carbon atoms, and includes methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl and t-butyl. C₁₋₆alkyl additionally includes pentyl,n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphaticisomers thereof. C₀₋₄alkyl and C₀₋₆alkyl additionally indicates that noalkyl group need be present (e.g., that a covalent bond is present).

Any C₁₋₄alkyl or C₁₋₆alkyl may be optionally substituted with the groupR^(X), which may be on any carbon atom that results in a stablestructure and is available by conventional synthetic techniques.Suitable groups for R^(X) are C₁₋₄alkyl, OR′, SR′, C₁₋₄alkylsulfonyl,C₁₋₄alkylsulfoxyl, arylsulfonyl, arylsulfoxyl, C₁₋₄alkyl sulfonamides,aryl sulfonamides, —CN, N(R′)₂, CH₂N(R′)₂, —NO₂, —CF₃, —CO₂R′—CON(R′)₂,—COR′, —NR′C(O)R′, F, Cl, Br, I, or CF₃S(O)_(r)—, wherein R′ and r areas defined for formula (I) compounds.

Halogen or halo means F, Cl, Br, and I.

Ar, or aryl, as applied herein, means phenyl or naphthyl, or phenyl ornaphthyl substituted by one to three substituents, such as those definedabove for alkyl, or substituted by methylenedioxy.

Het, or heterocycle, indicates an optionally substituted five or sixmembered monocyclic ring, or a nine or ten-membered bicyclic ringcontaining one to three heteroatoms chosen from the group of nitrogen,oxygen and sulfur, which are stable and available by conventionalchemical synthesis. Illustrative heterocycles are benzofuryl,benzimidazolyl, benzopyranyl, benzothienyl, furyl, imidazolyl,indolinyl, morpholinyl, piperidinyl, piperazinyl, pyrrolyl,pyrrolidinyle, tetrahydropyridinyl, pyridinyl, thiazolyl, thienyl,quinolinyl, isoquinolinyl, and tetra- and perhydro-quinolinyl andisoquinolinyl. Any accessible combination of up to three substituents onthe Het ring, such as those defined above for alkyl, that are availableby chemical synthesis and are stable are within the scope of thisinvention.

Certain radical groups are abbreviated herein. t-Bu refers to thetertiary butyl radical, Boc refers to the t-butyloxycarbonyl radical,Fmoc refers to the fluorenylmethoxycarbonyl radical, Ph refers to thephenyl radical, Cbz refers to the benzyloxycarbonyl radical, Bn refersto the benzyl radical, Me refers to methyl, Et refers to ethyl, Acrefers to acetyl, Alk refers to C₁₋₄alkyl, Nph refers to 1- or2-naphthyl and cHex refers to cyclohexyl. Tet refers to 5-tetrazolyl.

Certain reagents are abbreviated herein. DCC refers todicyclohexylcarbodiimide, DMAP refers to dimethylaminopyridine, EDCrefers to 1-(3-dimethylaminopropyl)-3-ethylcarbodilmide, hydrochloride,HOBt refers to 1-hydroxybenzotriazole, THF refers to tetrahydrofuran,DIEA refers to diisopropylethylamine, DEAD refers to diethylazodicarboxylate, PPh₃ refers to triphenylphosphine, DIAD refers todiisopropyl azodicarboxylate, DME refers to dimethoxyethane, DMF refersto dimethylformamide, NBS refers to N-bromosuccinimide, Pd/C refers to apalladium on carbon catalyst, PPA refers to polyphosphoric acid, DPPArefers to diphenylphosphoryl azide, BOP refers tobenzotriazol-1-yloxy-tris(dimethyl-amino)phosphoniumhexafluorophosphate, HF refers to hydrofluoric acid, TEA refers totriethylamine, TFA refers to trifluoroacetic acid, PCC refers topyridinium chlorochromate.

Generally, the compounds of formula (I) are prepared by reacting acompound of formula (II) with a compound of formula (III):

wherein R¹, R² and X are as defined in formula (I), with any reactivefunctional groups protected, in the presence of EDC and HOBT;

and thereafter removing any protecting groups, and optionally forming apharmaceutically acceptable salt.

In particular, compounds of formula (I) are prepared by the generalmethods described in Scheme I.

Reagents and conditions: (a) N-(benzyloxycarbonyl)oxysuccinimide, Et₃N,DMF; (b) 4-benzyloxybenzyl chloride, NaH, DMF; (c) H₂, 10% Pd/C, HCl,MeOH, dioxane, (d) 4-hydroxybenzoic acid, EDC, HOBt H₂O, (i-Pr)₂NEt,DMF.

Commercially available 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole (I-1)is protected at the piperidine nitrogen with a suitable protectinggroup, for instance a benzyloxycarbonyl (Cbz) group, to afford I-2. Theuse of protecting groups to mask reactive functionality is well-known tothose of skill in the art, and other protecting groups are listed instandard reference volumes, such as Greene, “Protective Groups inOrganic Synthesis” (published by Wiley-Interscience). The indolenitrogen is functionalized as appropriate with, for instance, alkylgroups, arylalkyl groups, acyl groups, or sulfonyl groups, to affordN-substituted derivatives. For example, the indole nitrogen can bealkylated with a suitable benzyl halide derivative, such as4-benzyloxybenzyl chloride, to afford the benzylated derivative I-3. Thealkylation reaction generally requires deprotonation of the indole witha strong base, such as sodium hydride, LDA, or LiN(TMS)₂, and istypically conducted in a polar, aprotic solvent, usually THF, DMF, ormixtures thereof. The Cbz protecting group is removed by hydrogenationunder acidic conditions, in the presence of a catalytic amount ofpalladium metal on activated carbon (Pd/C), to provide the aminehydrochloride I-4. Other standard methods for removal of a Cbzprotecting group are described by Greene (cited above). The aminederivative is then converted to amide I-5 by reaction with an activatedderivative of a suitable carboxylic acid. For example, 4-hydroxybenzoicacid is converted to an activated form by reaction with EDC and HOBt,and the activated form is subsequently reacted with amine I-4 in asuitable solvent such as DMF, CH₂Cl₂, or CH₃CN. Depending on whetheracid neutralization is required, an added base, such as triethylamine(Et₃N), diisopropylethylamine ((i-Pr)₂NEt), or pyridine, may be used.

Many additional methods for converting a carboxylic acid to an amide areknown, and can be found in standard reference books, such as “Compendiumof Organic Synthetic Methods”, Vol. I-VI (published byWiley-lnterscience), or Bodansky, “The Practice of Peptide Synthesis”(published by Springer-Verlag), which are incorporated herein byreference.

Amide coupling reagents as used herein denote reagents which may be usedto form peptide bonds. Typical coupling methods employ carbodiimides,activated anhydrides and esters and acyl halides. Reagents such as EDC,DCC, DPPA, PPA, BOP reagent, HOBt, N-hydroxysuccinimide and oxalylchloride are typical.

Typically, the amine is coupled via its free amino group to anappropriate carboxylic acid substrate using a suitable carbodiimidecoupling agent, such as N,N′ dicyclohexyl carbodiimide (DCC), optionallyin the presence of catalysts such as 1-hydroxybenzotriazole (HOBt) anddimethylamino pyridine (DMAP). Other methods, such as the formation ofactivated esters, anhydrides or acid halides, of the free carboxyl of asuitably protected acid substrate, and subsequent reaction with the freeamine, optionally in the presence of a base, are also suitable. Forexample, a benzoic acid is treated in an anhydrous solvent, such asmethylene chloride or tetrahydrofuran (THF), in the presence of a base,such as N-methylmorpholine, DMAP or a trialkylamine, with isobutylchloroformate to form the “activated anhydride”, which is subsequentlyreacted with the free amine.

Acid addition salts of the compounds are prepared in a standard mannerin a suitable solvent from the parent compound and an excess of an acid,such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric,acetic, trifluoroacetic, maleic, succinic or methanesulfonic. Certain ofthe compounds form inner salts or zwitterions which may be acceptable.Cationic salts are prepared by treating the parent compound with anexcess of an alkaline reagent, such as a hydroxide, carbonate oralkoxide, containing the appropriate cation; or with an appropriateorganic amine. Cations such as Li⁺, Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺ and NH₄ ⁺ arespecific examples of cations present in pharmaceutically acceptablesalts.

This invention also provides a pharmaceutical composition whichcomprises a compound according to formula (I) and a pharmaceuticallyacceptable carrier. Accordingly, the compounds of formula (I) may beused in the manufacture of a medicament. Pharmaceutical compositions ofthe compounds of formula (I) prepared as hereinbefore described may beformulated as solutions or lyophilized powders for parenteraladministration. Powders may be reconstituted by addition of a suitablediluent or other pharmaceutically acceptable carrier prior to use. Theliquid formulation may be a buffered, isotonic, aqueous solution.Examples of suitable diluents are normal isotonic saline solution,standard 5% dextrose in water or buffered sodium or ammonium acetatesolution. Such formulation is especially suitable for parenteraladministration, but may also be used for oral administration orcontained in a metered dose inhaler or nebulizer for insufflation. Itmay be desirable to add excipients such as polyvinylpyrrolidone,gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol,sodium chloride or sodium citrate.

Alternately, these compounds may be encapsulated, tableted or preparedin a emulsion or syrup for oral administration. Pharmaceuticallyacceptable solid or liquid carriers may be added to enhance or stabilizethe composition, or to facilitate preparation of the composition. Solidcarriers include starch, lactose, calcium sulfate dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. Liquid carriers include syrup, peanut oil, olive oil, salineand water. The carrier may also include a sustained release materialsuch as glyceryl monostearate or glyceryl distearate, alone or with awax. The amount of solid carrier varies but, preferably, will be betweenabout 20 mg to about 1 g per dosage unit. The pharmaceuticalpreparations are made following the conventional techniques of pharmacyinvolving milling, mixing, granulating, and compressing, when necessary,for tablet forms; or milling, mixing and filling for hard gelatincapsule forms. When a liquid carrier is used, the preparation will be inthe form of a syrup, elixir, emulsion or an aqueous or non-aqueoussuspension. Such a liquid formulation may be administered directly p.o.or filled into a soft gelatin capsule.

For rectal administration, the compounds of this invention may also becombined with excipients, such as cocoa butter, glycerin, gelatin orpolyethylene glycols, and molded into a suppository.

For topical administration, the compounds of this invention may becombined with diluents to take the form of ointments, gels, pastes,creams, powders or sprays. The compositions which are ointments, gels,pastes or creams contain diluents, for example, animal and vegetablefats, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures of these substances. The compositions which arepowders or sprays contain diluents, for example, lactose, talc, silicicacid, aluminum hydroxide, calcium silicate and polyamide powder, ormixtures of these substances. Additionally, for topical ophthalmologicadministration, the typical carriers are water, mixtures of water andwater miscible solvents, such as lower alkanols or vegetable oils, andwater-soluble non-toxic polymers, for example cellulose derivatives,such as methyl cellulose.

The compounds described herein are inhibitors of FabI, and are usefulfor treating bacterial infections. For instance, these compounds areuseful for the treatment of bacterial infections, such as, for example,infections of upper respiratory tract (e.g. otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.empyema, lung abscess), cardiac (e.g. infective endocarditis),gastrointestinal (e.g. secretory diarrhoea, splenic abscess,retroperitoneal abscess), CNS (e.g. cerebral abscess), eye (e.g.blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.epididymitis, intrarenal and perinephric abscess, toxic shock syndrome),skin (e.g. impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis), and bone and joint (e.g. septicarthritis, osteomyelitis). Also, the compounds of this invention may beuseful as antifungal agents. Additionally, the compounds may be usefulin combination with known antibiotics.

The compounds of this invention are administered to the patient, in amanner such that the concentration of drug is sufficient to treatbacterial infections. The pharmaceutical composition containing thecompound is administered at an oral dose of between about 10 mg to about1000 mg, taken once or several times daily, in a manner consistent withthe condition of the patient. Preferably, the oral dose would be about50 mg to about 500 mg, although the dose may be varied depending uponthe age, body weight and symptoms of the patient. For acute therapy,parenteral administration is preferred. An intravenous infusion of thecompound of formula (I) in 5% dextrose in water or normal saline, or asimilar formulation with suitable excipients, is most effective,although an intramuscular bolus injection is also useful. The preciselevel and method by which the compounds are administered is readilydetermined by one skilled in the art.

The compounds may be tested in one of several biological assays todetermine the concentration of compound which is required to have agiven pharmacological effect.

Cloning of S. aureus FabI:

The fabI gene was cloned from the chromosomal DNA of S. aureus strainWCUH29 using the polymerase chain reaction. Amplification was performedusing Taq DNA polymerase (BRL) and the following primers:

5′-CGCCTCGAGATGTFAAATCTTGAAAACAAAACATATGTC-3′ and

5′-CGCGGATCCAATCAAGTCAGGTTGAAATATCCA-3′ (XhoI and BamHI sitesunderlined). The resulting fragment was then digested with XhoI andBamHI and ligated into XhoI- and BamHI-digested expression vectorpET-16b (Novagen), producing pET-His₁₀-fabI. The gene sequence of fabIwas confirmed by automated cycle sequencing using an Applied Biosystemsmodel 377 machine. The untagged version of pET-fabI was constructed bydigesting pET-His₁₀-fabI with NcoI and NdeI to remove a 97 bp fragmentencoding the His 10 tag, the factor Xa cleavage site and the first 8amino acids of FabI, and replacing it with a linker encoding the first 8amino acids of FabI plus a glycine residue between the initiatormethionine and the lysine at position 2. This plasmid was calledpET-fabI. The linker was made by annealing the following twooligonucleotides:

5′-CATGGGCTTAAATCTTGAAAACAAAACA-3′ and

5′-TATGTTTTGTTTTCAAGATTTAAGCC-3′. The linker sequence in pET-fabI wasconfirmed by dideoxy sequencing. Only native FabI was used for compoundevaluation. For overproduction of native FabI, plasmid pET-fabI wastransformed into BL21(DE3) (Novagen) cells, to form strainBL21(DE3):pET-fabI.

Purification of S. aureus FabI

S. aureus FabI was expressed as soluble protein to 10% of total cellprotein, 400 g cells being recovered from 15L fermentation in tryptonephosphate medium. The cells were lysed and the sample centrifuged. Theresulting supernatant was filtered and purified using three consecutivechromatography columns: ion-exchange (Sourse 15Q), dye-affinity (Bluesepharose), and size exclusion chromatography columns (Superose 12).After each column the FabI containing fractions were pooled,concentrated, and checked for purity and biological activity.

Cloning of E. coli FabI:

A PCR fragment of correct size for E. coli Fabi was PCR amplified fromE. coli chromosomal DNA, subcloned into the TOPO TA cloning vector, andverified by colony PCR+restriction endonuclease analysis. Thepresumptive E. coli FabI PCR fragment was subcloned into the expressionvector pBluePet. The FabI clone was transformed into E. coli strainBL21(DE3). Small Scale expression studies show an over-expressed proteinband of correct molecular weight (˜28 Kda) for E. coli FabI clearlyvisible following Coomassie staining of SDS PAGE gels. DNA sequencing ofthe E. coli FabI expression constricts illustrated that no errors wereapparent. N′ terminal amino acid sequencing has confirmed theover-expressed protein band to be E. coli FabI.

Purification of E. coli FabI

E. coli FabI was expressed as soluble protein to 15% of total cellprotein, 120 g cells being recovered from 3L fermentation in shakeflasks in modified terrific broth. The cells were lysed and the samplecentrifuged. The resulting supernatant was filtered and purified usingthree consecutive chromatography columns: ion-exchange (Sourse 15Q),dye-affinity (blue sepharose), and size exclusion (superose 12). Aftereach column the FabI containing fractions were pooled, concentrated andchecked for purity and biological activity.

S aureus FabI Enzyme Inhibition Assay (NADH):

Assays were carried out in half-area, 96-well microtitre plates.Compounds were evaluated in 50-uL assay mixtures containing 100 mMNaADA, pH 6.5 (ADA=N-[2-acetamido]-2-iminodiacetic acid), 4% glycerol,0.25 mM crotonoyl CoA, 1 mM NADH, and an appropriate dilution of S.aureus FabI. Inhibitors were typically varied over the range of 0.01-10uM. The consumption of NADH was monitored for 20 minutes at 30° C. byfollowing the change in absorbance at 340 nm. Initial velocities wereestimated from an exponential fit of the non-linear progress curvesrepresented by the slope of the tangent at t=0 min. IC₅₀'s wereestimated from a fit of the initial velocities to a standard,4-parameter model and are typically reported as the mean ±S.D. ofduplicate determinations. Triclosan, a commercial antibacterial agentand inhibitor of FabI, is currently included in all assays as a positivecontrol. Compounds of this invention have IC₅₀'s from about 30.0micromolar to about 0.010 micromolar.

S aureus FabI Enzyme Inhibition Assay (NADPH):

Assays were carried out in half-area, 96-well microtitre plates.Compounds were evaluated in 15-uL assay mixtures containing 100 mMNaADA, pH 6.5 (ADA=N-[2-acetamido]-2-iminodiacetic acid), 4% glycerol,0.25 mM crotonoyl CoA, 50 uM NADPH, and an appropriate dilution of S.aureus FabI. Inhibitors were typically varied over the range of 0.01-10uM. The consumption of NADPH was monitored for 20 minutes at 30° C. byfollowing the change in absorbance at 340 nm. Initial velocities wereestimated from an exponential fit of the non-linear progress curvesrepresented by the slope of the tangent at t=0 min. IC₅₀'s wereestimated from a fit of the initial velocities to a standard,4-parameter model and are typically reported as the mean ±S.D. ofduplicate determinations. Triclosan, a commercial antibacterial agentand inhibitor of FabI, is currently included in all assays as a positivecontrol.

E. coli FabI Enzyme Inhibition Assay:

Assays were carried out in half-area, 96-well microtitre plates.Compounds were evaluated in 150-uL assay mixtures containing 100 mMNaADA, pH 6.5 (ADA=N-[2-acetamido]-2-iminodiacetic acid), 4% glycerol,0.25 mM crotonoyl CoA, 50 uM NADH, and an appropriate dilution of E.coli FabI. Inhibitors were typically varied over the range of 0.01-10uM. The consumption of NADH was monitored for 20 minutes at 30° C. byfollowing the change in absorbance at 340 nm. Initial velocities wereestimated from an exponential fit of the non-linear progress curvesrepresented by the slope of the tangent at t=0 min. IC₅₀'s wereestimated from a fit of the initial velocities to a standard,4-parameter model and are typically reported as the mean ±S.D. ofduplicate determinations. Triclosan, a commercial antibacterial agentand inhibitor of FabI, is currently included in all assays as a positivecontrol. Compounds of this invention have IC₅₀'s from about 45.0micromolar to about 2.0 micromolar.

Antimicrobial Activity Assay:

Whole-cell antimicrobial activity was determined by broth microdilutionusing the National Committee for Clinical Laboratory Standards (NCCLS)recommended procedure, Document M7-A4, “Methods for DilutionSusceptibility Tests for Bacteria that Grow Aerobically”. The compoundwas tested in serial two-fold dilutions ranging from 0.06 to 64 mcg/mL.Antibacterial activity is assessed by screening the compound againstrepresentative bacteria, selected from the following: Staphylococcusaureus Oxford, Staphylococcus aureus WCUH29, Streptococcus pneumoniaeR6, Streptococcus pneumoniae ER Y2, Streptococcus pneumoniae 1629,Streptococcus pneumoniae N 1387, Streptococcus pyogenes CN10,Enterococcus faecalis I, Enterococcus faecalis 7, Haemophilus influenzaeQ1 , Haemophilus influenzae NEMC1l, Moraxella Catarrhalis 1502,Escherichia coli DC0, Escherichia coli ESS, Escherichia coli 7623AcrAB+, Escherichia coli 120 AcrAB−, Escherichia coli MG1655,Escherichia coli MG1658, Klebsielia pneumoniae E70, Pseudomonasaeruginosa K799 wt. The minimum inhibitory concentration (MIC) wasdetermined as the lowest concentration of compound that inhibitedvisible growth. A mirror reader was used to assist in determining theMIC endpoint.

One skilled in the art would consider any compound with a MIC of lessthan 256 μg/mL to be a potential lead compound. Preferably, thecompounds used in the antimicrobial assays of the present invention havea MIC value of less than 128 μg/mL. Most preferably, said compounds havea MIC value of less than 64 μg/mL.

The examples which follow are intended in no way to limit the scope ofthis invention, but are provided to illustrate how to make and use thecompounds of this invention. Many other embodiments will be readilyapparent to those skilled in the art.

EXAMPLES General

Proton nuclear magnetic resonance (¹H NMR) spectra were recorded at 300MHz, and chemical shifts are reported in parts per million (delta)downfield from the internal standard tetramethylsilane (TMS).Abbreviations for NMR data are as follows: s=singlet, d=doublet,t=triplet, q=quartet, m=multiplet, dd=doublet of doublets, dt=doublet oftriplets, app=apparent, br=broad. J indicates the NMR coupling constantmeasured in Hertz. CDCl₃ is deuteriochloroform, DMSO-d₆ ishexadeuteriodimethylsulfoxide, and CD₃OD is tetradeuteriomethanol. Massspectra were obtained using electrospray (ES) ionization techniques.Elemental analyses were performed by Quantitative Technologies Inc.,Whitehouse, N.J. Melting points were obtained on a Thomas-Hoover meltingpoint apparatus and are uncorrected. All temperatures are reported indegrees Celsius. Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254thin layer plates were used for thin layer chromatography. Flashchromatography was carried out on E. Merck Kieselgel 60 (230-400 mesh)silica gel. Analytical HPLC was performed on Beckman chromatographysystems. Preparative HPLC was performed using Gilson chromatographysystems. ODS refers to an octadecylsilyl derivatized silica gelchromatographic support. YMC ODS-AQ® is an ODS chromatographic supportand is a registered trademark of YMC Co. Ltd., Kyoto, Japan. PRP-1® is apolymeric (styrene-divinylbenzene) chromatographic support, and is aregistered trademark of Hamilton Co., Reno, Nev. Celite® is a filter aidcomposed of acid-washed diatomaceous silica, and is a registeredtrademark of Manville Corp., Denver, Colo.

Preparation 1 Preparation of9-[4-(Hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indoleMono Hydrochloride

a) 2-Benzyloxycarbonyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

To a stirred solution of 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole (20.0g, 116.2 mmole) in dry DMF (150 mL) at RT was addedN-(benzyloxycarbonyloxy)succinimide (31.84 g, 127.76 mmole) andtriethylamine (13.0 g, 127.76 mmole). After 12 h, the reaction contentswere poured into H₂O (150 mL) and extracted with EtOAc (2×200 mL). Thecombined organic phases were washed sequentially with H₂O and brine,then were dried over Na₂SO₄. Concentration under reduced pressure gave ayellow oil. Purification on silica (hexanes/EtOAc, 4:1) afforded thetitle compound (34.48 g, 97%) as a white solid: MS (ES) m/e 307 (M+H)⁺.

b)2-Benzyloxycarbonyl-9-[(4-benzyloxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

To a stirred solution of2-benzyloxycarbonyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole (17.75 g,58.0. mmole) in dry DMF (150 mL) at was added 4-benzyloxybenzylchloride(14.85 g, 63.8 mmole). After 10 min, 60% NaH (2.78 g, 69.6 mmole) wasadded and the reaction slurry was allowed to warm to RT and stir for 12hr. The reaction contents were poured into H₂O (200 mL) and extractedwith EtOAc (2×200 mL). The combined organic phases were washedsequentially with H₂O and brine, then were dried over Na₂SO₄.Concentration under reduced pressure gave a waxy solid. Purification onsilica (hexanes/EtOAc, 4:1) afforded the title compound (27.13 g, 93%)as a white solid: MS (ES) m/e 503 (M+H)⁺.

c) 9-[4-(Hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indoleMono Hydrochloride

To a solution of2-benzyloxycarbonyl-9-[(4-benzyloxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4b]indole(5.0 g, 9.96 mmole) in methanol (25 mL) and dioxane (50 mL) containingHCl (10 mL, 1M in dioxane) at RT in a Parr hydrogenation flask was added10% Pd/C (0.5 g). The reaction mixture was shaken under 45 psi of H₂ for5 hr. The suspension was filtered through celite®, and the filter padwas washed with methanol. The filtrate was concentrated on the rotavap,and the residue was dried under high vacuum to afford the titledcompound (2.79 g, 89%) as a white solid: MS (ES) m/e 279 (M+H−HCl)⁺.

Preparation 2 Preparation of9-Methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-indole Mono Hydrochloride

a)2-Benzyloxycarbonyl-9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

To a stirred solution of2-benzyloxycarbonyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole (5.0 g,16.3 mmole) in dry DMF (75 mL) at 5° C. was added 60% NaH (0.7 g, 17.5mmole). After 10 min, methyliodide (11.57 g, 81.5 mmole) was added andthe reaction was allowed to warm to RT and stir for 12 hr. The reactioncontents were poured into H₂O (100 mL) and extracted with EtOAc (2×100mL). The combined organic phases were washed sequentially with H₂O andbrine, then were dried over Na₂SO₄. Concentration under reduced pressureand purification on silica (hexanes/EtOAc, 1:1) afforded the titlecompound (5.17 g, 99%) as a white solid: MS (ES) m/e 321 (M+H)⁺.

b) 9-Methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-indole MonoHydrochloride

To a solution of2-benzyloxycarbonyl-9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole(5.17 g, 16.2 mmole) in methanol (100 mL), EtOAc (25 ml) and HCl (17 mL,1M in dioxane) at RT in a Parr hydrogenation flask was added 10% Pd/C(0.5 g). The reaction mixture was shaken under 45 psi of H₂ for 6 hr.The suspension was filtered through celite®, and the filter pad waswashed with methanol. The filtrate was concentrated on the rotavap, andthe residue was dried under high vacuum to afford the titled compound(3.19 g, 92%) as a white solid: MS (ES) m/e 187 (M+H−HCl)⁺.

Preparation 3 Preparation of9-{[4-(trifluoromethyl)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4b]-indoleMono Hydrochloride

a)2-Benzyloxycarbonyl-9-{[4-(trifluoromethyl)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

To a stirred solution of2-benzyloxycarbonyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole (5.82 g,19.0 mmole) in dry DMF (75 mL) at 5° C. was added 60% NaH (1.10 g, 28.5mmole). After 10 min, 4-trifluoromethylbenzyl bromide (5.0 g, 20.9mmole) was added and the reaction was allowed to warm to RT and stir for12 hr. The reaction contents were poured into H₂O (100 mL) and extractedwith EtOAc (2×100 mL). The combined organic phases were washedsequentially with H₂O and brine, thenwere dried over Na₂SO₄.Concentration under reduced pressure and purification on silica(hexanes/EtOAc, 4:1) afforded the title compound (8.73 g, 99%) as awhite solid: MS (ES) m/e 465 (M+H)⁺.

b)9-{[4-(Trifluoromethyl)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-indoleMono Hydrochloride

To a solution of2-benzyloxycarbonyl-9-{[4-(trifluoromethyl)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole(8.73 g, 18.81 mmole) in methanol (50 mL), dioxane (50 mL) and HCl (19mL, 1M in dioxane) at RT in a Parr hydrogenation flask was added 10%Pd/C (0.5 g). The reaction mixture was shaken under 45 psi of H₂ for 6hr. The suspension was filtered through celite®, and the filter pad waswashed with methanol. The filtrate was concentrated on the rotavap, andthe residue was dried under high vacuum to afford the titled compound(6.33 g, 92%) as a white solid: MS (ES) m/e 331 (M+H−HCl)⁺.

Preparation 4 Preparation of2-Benzoyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a) 2-Benzoyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

To a stirred solution of 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole (2.0g, 11.6 mmole) in dry DMF (20 mL) at RT was added benzoic acid (1.56 g,12.77 mmole), 1-hydroxybenzotriazole hydrate (1.72 g, 12.77 mmole) anddiisopropylethylamine (1.65 g, 12.77 mmole). After 10 min, EDC (2.44 g,12.77 mmole) was added and the reaction was allowed to stir for 12 hr.The reaction contents were poured into H₂O (100 mL) and extracted withEtOAc (2×100 mL). The combined organic phases were washed sequentiallywith H₂O and brine, then were dried over Na₂SO₄. Concentration underreduced pressure and purification on silica (hexanes/EtOAc, 1:1)afforded the title compound (3.0 g, 94%) as a white solid: MS (ES) m/e277 (M+H)⁺.

Preparation 5 Preparation of9-{[4-(Fluoro)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-indoleMono Hydrochloride

a)2-Benzyloxycarbonyl-9-{[4-(fluoro)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

To a stirred solution of2-benzyloxycarbonyl-1,3,4,9-tetrahydro-2H-pyrido[3,4 b]indole (5.0 g,16.33 mmole) in dry DMF (75 mL) at 5° C. was added 60% NaH (0.98 g, 24.5mmole). After 10 min, 4-fluorobenzyl bromide 3,40 g, 18.0 mmole) wasadded and the reaction was allowed to warm to RT and stir for 12 hr. Thereaction contents were poured into H₂O (100 mL) and extracted with EtOAc(2×100 mL). The combined organic phases were washed sequentially withH₂O and brine, then were dried over Na₂SO₄. Concentration under reducedpressure and purification on silica (hexanes/EtOAc, 4:1) afforded thetitle compound (6.42 g, 95%) as a white solid: MS (ES) m/e 415 (M+H)⁺.

b)9-{[4-(Fluoro)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-indoleMono Hydrochloride

To a solution of2-benzyloxycarbonyl-9-{[4-(fluoro)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole(5.28 g, 12.7 mmole) in methanol (50 mL), dioxane (50 mL) and HCl (19mL, 1M in dioxane) at RT in a Parr hydrogenation flask was added 10%Pd/C (0.5 g). The reaction mixture was shaken under 45 psi of H₂ for 6hr. The suspension was filtered through celite®, and the filter pad waswashed with methanol. The filtrate was concentrated on the rotavap, andthe residue was dried under high vacuum to afford the titled compound(3.61 g, 90%) as a white solid: MS (ES) m/e 281 (M+H−HCl)⁺.

Preparation 6 Preparation of Methyl4-(1,2,3,4-Tetrahydrobeta-carbolin-9-yl}methyl)benzoate MonoHydrochloride

a)2-Benzyloxycarbonyl-9-{[4-(carboxymethyl)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

To a stirred solution of2-benzyloxycarbonyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole (5.0 g,16.33 mmole) in dry DMF (75 mL) at 5° C. was added 60% NaH (0.98 g, 24.5mmole). After 10 min, methyl 4-(bromomethyl)benzoate (4.12 g, 18.0mmole) was added and the reaction was allowed to warm to RT and stir for12 hr. The reaction contents were poured into H₂O (100 mL) and extractedwith EtOAc (2×100 mL). The combined organic phases were washedsequentially with H₂O and brine, then were dried over Na₂SO₄.Concentration under reduced pressure and purification on silica(hexanes/EtOAc, 4:1) afforded the title compound (6.91 g, 93%) as awhite solid: MS (ES) m/e 455 (M+H)⁺.

b)9-{[4-(Carboxymethyl)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-indoleMono Hydrochloride

To a solution of2-benzyloxycarbonyl-9-{[4-(carboxymethyl)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole(6.91 g, 15.22 mmole) in methanol (50 mL), dioxane (50 mL) and HCl (19mL, 1M in dioxane) at RT in a Parr hydrogenation flask was added 10%Pd/C (0.5 g). The reaction mixture was shaken under 45 psi of H₂ for 6hr. The suspension was filtered through celite®, and the filter pad waswashed with methanol. The filtrate was concentrated on the rotavap, andthe residue was dried under high vacuum to afford the titled compound(5.22 g, 90%) as a white solid: MS (ES) m/e 345 (M+H−HCl)⁺.

The following examples illustrate methods for preparing the biologicallyactive compounds of this invention from intermediate compounds such asthose described in the foregoing Preparations.

Example 1 Preparation of2-Benzoyl-9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a) 2-Benzoyl-9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

To a stirred solution of2-benzoyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole (3.0 g, 10.9 mmole)in dry DMF (75 mL) at 5° C. was added 60% NaH (0.52 g, 13.1 mmole).After 10 min, methyliodide (7.80 g, 55 mmole) was added and the reactionwas allowed to warm to RT and stir for 12 hr. The reaction contents werepoured into H₂O (50 mL) and extracted with EtOAc (2×100 mL). Thecombined organic phases were washed sequentially with H₂O and brine,then were dried over Na₂SO₄. Concentration under reduced pressure andpurification on silica (hexanes/EtOAc, 1:1) afforded the title compound(3.14 g, 99%) as a white solid: MS (ES) m/e 291 (M+H)⁺.

Example 2 Preparation of2-Benzoyl-9-[(4-benzyloxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-prido[3,4-b]indole

a)2-Benzoyl-9-[(4-benzyloxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

To a stirred solution of2-benzoyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole (17.75 g, 58.0mmole) in dry DMF (75 mL) at 5° C. was added 60% NaH (2.78 g, 69.6mmole). After 10 min, 4-benzyloxybenzyl chloride (14.85 g, 63.8 mmole)was added and the reaction was allowed to warm to RT and stir for 12 hr.The reaction contents were poured into H₂O (150 mL) and extracted withEtOAc (2×150 mL). The combined organic phases were washed sequentiallywith H₂O and brine, then were dried over Na₂SO₄. Concentration underreduced pressure and purification on silica (hexanes/EtOAc, 1:1)afforded the title compound (26.61 g, 97%) as a white solid: MS (ES) m/e473 (M+H)⁺.

Example 3 Preparation of2-(3-Amino)benzoyl-9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-indole

a)2-[3-(Amino)benzoyl]-9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-indole(385820)

To a stirred solution of9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-indole mono hydrochloride(0.35 g, 1.59 mmole) in dry DMF (20 mL) at RT was added 3-aminobenzoicacid (0.24 g, 1.75 mmole), 1-hydroxybenzotriazole hydrate (0.24 g, 1.75mmole) and diisopropylethylamine (0.45 g, 3.50 mmole). After 10 min, EDC(0.33 g, 1.75 mmole) was added and the reaction was allowed to stir for12 hr. The reaction contents were poured into H₂O (100 mL) and extractedwith EtOAc (2×100 mL). The combined organic phases were washedsequentially with H₂O and brine, then were dried over Na₂SO₄.Concentration under reduced pressure gave a yellow oil. Purification onsilica (CHC13/CH3OH, 95:5) afforded the title compound (0.45 g, 92%) asa white solid: MS (ES) m/e 306 (M+H)⁺.

Example 4 Preparation of2-(4-Hydroxy)benzoyl-9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a)2-(4-Hydroxy)benzoyl-9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

To a stirred solution of9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4b]-indolemonohydrochloride (0.50 g, 1.59 mmole) in dry DMF (25 mL) at RT wasadded 4-hydroxybenzoic acid (0.24 g, 1.75 mmole), 1-hydroxybenzotriazolehydrate (0.24 g, 1.75 mmole) and diisopropylethylamine (0.45 g, 3.50mmole). After 10 min, EDC (0.33 g, 1.75 mmole) was added and thereaction was allowed to stir for 12 hr. The reaction contents werepoured into H₂O (100 mL) and extracted with EtOAc (2×150 mL). Thecombined organic phases were washed sequentially with H₂O and brine,then were dried over Na₂SO₄. Concentration under reduced andpurification on silica (hexanes/EtOAc, 1:2) afforded the title compound(0.55 g, 91%) as a white solid: MS (ES) m/e 383 (M+H)⁺.

Example 5 Preparation of2-Benzoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a)2-Benzoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting benzoicacid (0.21 g, 1.75 mmole) for 4-hydroxybenzoic acid, the title compound(0.60 g, 90%) was prepared as a white solid following flashchromatography on silica gel (hexanes/EtOAc, 1:2): MS (ES) m/e 383(M+H)⁺.

Example 6 Preparation of2-(3,4-Methylenedioxy)benzoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a)2-(3,4-Methylenedioxy)benzoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting piperonylicacid (0.29 g, 1.75 mmole) for 4-hydroxybenzoic acid, the title compound(0.69 g, 93%) was prepared as a white solid following flashchromatography on silica gel (hexanes/EtOAc, 1:2): MS (ES) m/e 427(M+H)⁺.

Example 7 Preparation of2-(2-Hydroxy)benzoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a)2-(2-Hydroxy)benzoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting2-hydroxybenzoic acid (0.24 g, 1.75 mmole) for 4-hydroxybenzoic acid,the title compound (0.62 g, 89%) was prepared as a white solid followingflash chromatography on silica gel (hexanes/EtOAc, 1:2): MS (ES) m/e 399(M+H)⁺.

Example 8 Preparation of2-(3-Imidazo)benzoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a)2-(3-Imidazo)benzoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting5-benzimidazolecarboxylic acid (0.28 g, 1.75 mmole) for 4-hydroxybenzoicacid, the title compound (0.63 g, 85%) was prepared as an off-whitesolid following flash chromatography on silica gel (hexanes/EtOAc, 1:2):MS (ES) m/e 423 (M+H)⁺.

Example 9 Preparation of2-(6-Hydroxy)nicotinoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a)2-(6-Hydroxy)nicotinoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting6-hydroxynicotinic acid (0.24 g, 1.75 mmole) for 4-hydroxybenzoic acid,the title compound (0.58 g, 83%) was prepared as an off-white solidfollowing flash chromatography on silica gel (hexanes/EtOAc, 1:2): MS(ES) m/e 400 (M+H)⁺.

Example 10 Preparation of2-(6-Amino)nicotinoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4b]indole

a)2-(6-Amino)nicotinoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting6-aminonicotinic acid (0.24 g, 1.75 mmole) for 4-hydroxybenzoic acid,the title compound (0.60 g, 87%) was prepared as anoff-white solidfollowing flash chromatography on silica gel (hexanes/EtOAc, 1:2): MS(ES) m/e 399 (M+H)⁺.

Example 11 Preparation of2-(4-Methylamino)benzoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a)2-(4-Methylamino)benzoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting4-methylaminobenzoic acid (0.26 g, 1.75 mmole) for 4-hydroxybenzoicacid, the title compound (0.61 g, 85%) was prepared as an off-whitesolid following flash chromatography on silica gel (hexanes/EtOAc, 1:2):MS (ES) m/e 412 (M+H)⁺.

Example 12 Preparation of2-(4-Amino)benzoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a)2-(4-Amino)benzoyl-9-[4-(hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting4-aminobenzoic acid (0.24 g, 1.75 mmole) for 4-hydroxybenzoic acid, thetitle compound (0.56 g, 80%) was prepared as an off-white solidfollowing flash chromatography on silica gel (hexanes/EtOAc, 1:2): MS(ES) m/e 398 (M+H)⁺.

Example 13 Preparation of2-(4-Chloro)benzoyl-9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a)2-(4-Chloro)benzoyl-9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting4-chlorobenzoic acid (0.27 g, 1.75 mmole) for 4-hydroxybenzoic acid, thetitle compound (0.63 g, 96%) was prepared as white solid following flashchromatography on silica gel (hexanes/EtOAc, 1:2): MS (ES) m/e 417(M+H)⁺.

Example 14 Preparation of2-(3,5-Dichloro-4-hydroxy)benzoyl-9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a)2-(3,5-Dichloro-4-hydroxy)benzoyl-9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting3,5-dichloro-4-hydroxybenzoic acid (0.36 g, 1.75 mmole) for4-hydroxybenzoic acid, the title compound (0.70 g, 94%) was prepared aswhite solid following flash chromatography on silica gel (hexanes/EtOAc,1:2): MS (ES) m/e 468 (M+H)⁺.

Example 15 Preparation of2-(3-Chloro-4-hydroxy)benzoyl-9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a)2-(3-Chloro-4-hydroxy)benzoyl-9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting3-chloro-4-hydroxybenzoic acid hemihydrate (0.32 g, 1.75 mmole) for4-hydroxybenzoic acid, the title compound (0.62 g, 91%) was prepared aswhite solid following flash chromatography on silica gel (hexanes/EtOAc,1:2): MS (ES) m/e 434 (M+H)⁺.

Example 16 Preparation of2-(4-Hydroxy)benzoyl-9-{[4-(trifluoromethyl)phenyl]methy}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

a)2-(4-Hydroxy)benzoyl-9-{[4-(trifluoromethyl)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting9-{[4-(trifluoromethyl)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-indolemonohydrochloride (0.50 g, 1,37 mmole) for9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-indolemonohydrochloride, the title compound (0.55 g, 90%) was prepared aswhite solid following flash chromatography on silica gel (hexanes/EtOAc,1:2): MS (ES) m/e 451 (M+H)⁺.

Example 17 Preparation of Methyl4-({2-[(4-Hydroxyphenyl)carbonyl]-1,2,3,4-tetrahydrobeta-carbolin-9-yl}methyl)benzoate

According to the procedure of Example 4, except substituting9-{[4-(carboxymethyl)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]-indolemono hydrochloride (0.76 g, 2.0 mmole) for9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido-[3,4-b]-indolemono hydrochloride, the title compound (0.87 g, 94%) was prepared as awhite solid following flash chromatography on silica gel (hexanes/EtOAc,1:2): MS (ES) m/e 466 (M+H)⁺.

Example 18 Preparation of2-(4-Chloro-2-hydroxy)benzoyl-9-[(4-hydroxyphenyl)methyl]1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting4-chloro-2-hydroxybenzoic acid (0.19 g, 1.10 mmole) for 4-hydroxybenzoicacid, the title compound (0.44 g, 95%) was prepared as a white solidfollowing flash chromatography on silica gel (hexanes/EtOAc, 1:2): MS(ES) m/e 469 (M+H)⁺.

Example 19 Preparation of2-(5-Chloro-2-hydroxy)benzoyl-9-[(4-hydroxyphenyl)methyl]1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting5-chloro-2-hydroxybenzoic acid (0.19 g, 1.10 mmole) for 4-hydroxybenzoicacid, the title compound (0.44 g, 94%) was prepared as a white solidfollowing flash chromatography on silica gel (hexanes/EtOAc, 1:2): MS(ES) m/e 469 (M+H)⁺.

Example 20 Preparation of2-(4-Hydroxy)benzoyl-9-[(4-fluorophenyl)methyl]1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting9-[(4-fluorophenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido-[3,4-b]indolemono hydrochloride (0.50 g, 1.58 mmole) for9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido-[3,4-b]indolemono hydrochloride, the title compound (0.58 g, 92%) was prepared as awhite solid following flash chromatography on silica gel (hexanes/EtOAc,1:1): MS (ES) m/e 401 (M+H)⁺.

Example 21 Preparation of2-(3-Chloro-4-hydroxy)benzoyl-9-[(4-fluorophenyl)methyl]1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting9-[(4-fluorophenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido-[3,4-b]indolemono hydrochloride (0.50 g, 1.58 mmole) for9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido-[3,4-b]indolemono hydrochloride and substituting 3-chloro4-hydroxybenzoic acid hemihydrate (0.32 g, 1.75 mmole) for 4-hydroxybenzoic acid, the titlecompound (0.60 g, 95%) was prepared as a white solid following flashchromatography on silica gel (hexanes/EtOAc, 1:1): MS (ES) m/e 436(M+H)⁺.

Example 22 Preparation of2-(3,5-Dichloro-4-hydroxy)benzoyl-9-[(4-fluorophenyl)methyl]1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting9-[(4-fluorophenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido-[3,4-b]indolemono hydrochloride (0.50 g, 1.58 mmole) for9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido-[3,4-b]indolemono hydrochloride and substituting 3,5-dichloro-4-hydroxybenzoic acid(0.36 g, 1.75 mmole) for 4-hydroxybenzoic acid, the title compound (0.68g, 92%) was prepared as a white solid following flash chromatography onsilica gel (hexanes/EtOAc, 1:1): MS (ES) m/e 470 (M+H)⁺.

Example 23 Preparation of2-(3-Chloro-4-hydroxy)benzoyl-9-{[4-(trifluoromethyl)phenyl]-methyl}1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting9-{[4-(trifluoromethyl)phenyl]methyl}-1,3,4,9-tetrahydro-2H-pyrido-[3,4-b]indolemono hydrochloride (0.50 g, 1,37 mmole) for9-[(4-hydroxyphenyl)methyl]-1,3,4,9-tetrahydro-2H-pyrido-[3,4-b]indolemono hydrochloride and substituting 3-chloro-4-hydroxybenzoic acid hemihydrate (0.27 g, 1.51 mmole) for 4-hydroxybenzoic acid, the titlecompound (0.56 g, 90%) was prepared as a white solid following flashchromatography on silica gel (hexanes/EtOAc, 1:1): MS (ES) m/e 453(M+H)⁺.

Example 24 Preparation of2-(2-Hydroxy-4-methyl)benzoyl-9-[(4-hydroxyphenyl)methyl]1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole

According to the procedure of Example 4, except substituting2-hydroxy-4-methylbenzoic acid (0.27 g, 1.75 mmole) for 4-hydroxybenzoicacid, the title compound (0.62 g, 95%) was prepared as a white solidfollowing flash chromatography on silica gel (hexanes/EtOAc, 1:2): MS(ES) m/e 413 (M+H)⁺.

Example 25 Parenteral Dosage Unit Composition

A preparation which contains 20 mg of the compound of Example 1 as asterile dry powder is prepared as follows: 20 mg of the compound isdissolved in 15 mL of distilled water. The solution is filtered understerile conditions into a 25 mL multi-dose ampoule and lyophilized. Thepowder is reconstituted by addition of 20 mL of 5% dextrose in water(D5W) for intravenous or intramuscular injection. The dosage is therebydetermined by the injection volume. Subsequent dilution may be made byaddition of a metered volume of this dosage unit to another volume ofD5W for injection, or a metered dose may be added to another mechanismfor dispensing the drug, as in a bottle or bag for IV drip infusion orother injection-infusion system.

Example 26 Oral Dosagte Unit Composition

A capsule for oral administration is prepared by mixing and milling 50mg of the compound of Example 1 with 75 mg of lactose and 5 mg ofmagnesium stearate. The resulting powder is screened and filled into ahard gelatin capsule.

Example 27 Oral Dosage Unit Composition

A tablet for oral administration is prepared by mixing and granulating20 mg of sucrose, 150 mg of calcium sulfate dihydrate and 50 mg of thecompound of Example 1 with a 10% gelatin solution. The wet granules arescreened, dried, mixed with 10 mg starch, 5 mg talc and 3 mg steaficacid; and compressed into a tablet.

The above description fully discloses how to make and use the presentinvention. However, the present invention is not limited to theparticular embodiments described hereinabove, but includes allmodifications thereof within the scope of the following claims. Thevarious references to journals, patents and other publications which arecited herein comprises the state of the art and are incorporated hereinby reference as though fully set forth.

What is claimed is:
 1. A compound according to formula (I):

wherein: R¹ is benzimidazolyl or pyridinyl, unsubstituted or substituted by C₁₋₄alkyl, OR′, N(R′)₂, F, Cl, Br, I, and CF₃, in which R′ is H or C₁₋₆alkyl; R² is H, C₁₋₆alkyl or Ar—C₀₋₆alkyl; X is H, C₁₋₄alkyl, OR′, SR′, C₁₋₄alkylsulfonyl, C₁₋₄alkylsulfoxyl, CN, N(R′)₂, CH₂N(R′)₂, NO₂, CF₃, CO₂R′, CON(R′)₂, COR′, NR°C(O)R′, F, Cl, Br, I, or CF₃S(O)_(r)—; R′ is H, C₁₋₆alkyl or Ar—C₀₋₆alkyl; and r is 0, 1 or 2; or a pharmaceutically acceptable salt thereof.
 2. A pharmaceutical composition which comprises a compound according to claim 1 and a pharmaceutically acceptable carrier.
 3. A method for inhibiting FabI which comprises administering to a subject in need thereof an effective amount of a compound according to claim
 1. 4. A method of treating bacterial infections which comprises administering to a subject in need thereof an effective amount of a compound according to claim
 1. 